The long-term objective of the proposed project is to enhance Dr. Talcott's skills in studying mechanisms of action and molecular biology of the anticarcinogenic activities of selected botanicals in breast cancer. This will greatly strengthen her ongoing research on the efficacy and safety of botanical compounds. MENTORED RESEARCH in Dr. Safe's laboratory: Dr. Talcott's preliminary data show that betulinic acid (BA) decreases the expression of oncogenic transcription factors Spl, 3 and 4 in ER-negative breast cancer cells. This response was accompanied by a decrease in microRNA 27a expression and increased levels of the Sp-protein suppressor ZBTB10 in breast cancer cells. In AIM 1 the effects of BA and derived compounds on ER-negative and ER-positive breast cancer cell growth and apoptosis will be investigated using techniques including western-blotting, real time and semiquantitative RT PCR, as well as cellular imaging. In AIM 2 the effects of BA and derivatives on proteasome-dependent and independent degradation of Sp-proteins and the role for this pathway in mediating the cytotoxicity of these compounds will be investigated. Additionally the mechanisms associated with the transcriptional repression of Spl, Sp3, and Sp4 will also focus on the role of rm'RNA 27a and it's interaction with ZBTB10. In AIM 3 the in vivo anticarcinogenic activity of BA and related compounds will be investigated using the athymic nude mouse xenograft model. Tumors from treated and untreated animals will be carefully analyzed for response-specific expression of genes/proteins observed in breast cancer cells treated with the same BA-derived compounds used in Aims 1 and 2. Pharmacometric analysis will be performed for the determination of the efficacy of BA and related compounds with the long-term goal of developing dosing regimens. Additionally, in vivo imaging will be performed for monitoring the effects of BA and related compounds on tumor-development. CAREER DEVELOPMENT PLAN: Dr. Talcott's knowledge in the areas of toxicology and molecular oncology will also be enhanced through coursework and her laboratory expertise in mechanistic molecular techniques will be increased by an integrative mentored research plan, that will provide a strong foundation for Dr. Talcott to progress as independent investigator. The proposed research is highly relevant to public health. It will investigate the anticarcinogenic properties of selected botanical compounds and determine underlying mechanisms of action and efficacy for treatment of breast cancer.